LOCUS Exported 6439 bp ds-DNA circular SYN 27-JAN-2022 DEFINITION Protein expression in bacterial cells. Coiled-coil domains, M1-L275.. ACCESSION . VERSION . KEYWORDS STX4AA-c005 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6439) TITLE Gileadi lab plasmids 2021 REFERENCE 2 (bases 1 to 6439) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 27, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6439 /organism="synthetic DNA construct" /mol_type="other DNA" RBS 12..34 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 54..71 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1212..1256 /codon_start=1 /product="peptide tag that allows for enzymatic biotinylation" /label=AviTag(TM) /translation="GLNDIFEAQKIEWHE" CDS 1257..1274 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1327..1345) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1341..1388 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1425..1880 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1512..1531) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1722..1743 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1972..2787) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 2695..2714 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2779..2798 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2909..3497 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3398..3417 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3651..3668 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3683..3822 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3748..3767) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3908..3930 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3924..4115) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4924..6006) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 5968..5987 /label=LacI-R /note="LacI, reverse primer" promoter complement(6007..6084) /gene="lacI" /label=lacI promoter primer_bind 6290..6309 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6393..6412 /label=T7 /note="T7 promoter, forward primer" promoter 6393..6411 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 6412..6436 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." ORIGIN 1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgtgcagc agccatcatc 61 atcatcatca cggcagcggc agcggctctg accaggaggc aaaaccttca actgaggact 121 tgggggataa gaaggaaggt gaatatatta aactcaaagt cattggacag gatagcagtg 181 agattcactt caaagtgaaa atgacaacac atctcaagaa actcaaagaa tcatactgtc 241 aaagacaggg tgttccaatg aattcactca ggtttctctt tgagggtcag agaattgctg 301 ataatcatac tccaaaagaa ctgggaatgg aggaagaaga tgtgattgaa gtttaccagg 361 agcaaacggg aggtatgcgg gacaggaccc acgagctgag acagggggat gacagctcgg 421 acgaagagga caaggagcgg gtcgcgctgg tggtgcaccc gggcacggca cggctgggga 481 gcccggacga ggagttcttc cacaaggtcc ggacaattcg gcagactatt gtcaaactgg 541 ggaataaagt ccaggagttg gagaaacagc aggtcaccat cctggccacg ccccttcccg 601 aggagagcat gaagcaggag ctgcagaacc tgcgcgatga gatcaaacag ctggggaggg 661 agatccgcct gcagctgaag gccatagagc cccagaagga ggaagctgat gagaactata 721 actccgtcaa cacaagaatg agaaaaaccc agcatggggt cctgtcccag caattcgtgg 781 agctcatcaa caagtgcaat tcaatgcagt ccgaataccg ggagaagaac gtggagcgga 841 ttcggaggca gctgaagatc accaatgctg ggatggtgtc tgatgaggag ttggagcaga 901 tgctggacag tgggcaaagc gaggtgtttg tgtccaatat cctgaaggac acgcaggtga 961 ctcgacaggc cttaaatgag atctcggccc ggcacagtga gatccagcag cttgaacgca 1021 gtattcgtga gctgcacgac atattcactt ttctggctac cgaagtggag atgcaggggg 1081 agatgatcaa tcggattgag aagaacatcc tgagctcagc ggactacgtg gaacgtgggc 1141 aggagcacgt caagacggcc ctggagaacc agaagaaggc gaggaagaag aaagtcttgt 1201 gaagcggtgg cggtctgaac gacatcttcg aggctcagaa aatcgaatgg cacgaacacc 1261 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg 1321 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 1381 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg 1441 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 1501 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 1561 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 1621 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 1681 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 1741 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 1801 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 1861 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt 1921 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa 1981 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 2041 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 2101 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 2161 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 2221 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg 2281 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc 2341 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg 2401 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa 2461 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt 2521 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac 2581 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg 2641 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg 2701 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca 2761 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga 2821 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 2881 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2941 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 3001 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 3061 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 3121 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 3181 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3241 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3301 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3361 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3421 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3481 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3541 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 3601 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 3661 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 3721 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3781 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 3841 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 3901 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 3961 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 4021 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 4081 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 4141 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 4201 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 4261 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta 4321 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag 4381 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt 4441 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt 4501 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg 4561 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg 4621 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 4681 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 4741 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 4801 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 4861 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg 4921 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 4981 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag 5041 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg 5101 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat 5161 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg 5221 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag 5281 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat 5341 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt 5401 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc 5461 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg 5521 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac 5581 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat 5641 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 5701 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 5761 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 5821 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 5881 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 5941 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 6001 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 6061 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 6121 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg 6181 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc 6241 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg 6301 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg 6361 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg 6421 agcggataac aattcccct //